In recent years a number of immunoassay techniques has been developed for the measurement of clinically important ligands. Typically, a competitive binding immunoassay utilizes a conjugate of a labeling substance and a binding component which participates in a binding reaction to produce two species of labeled complexes a bound species and a free species. The relative amounts of the labeled complexes are a function of the amount of the ligand to be detected in the test sample.
Where the labeling substance in the bound species and in the free species are substantially indistinguishable by the means used to measure the labeling substance, the bound and the free species must be physically separated. This type of assay is referred to as heterogeneous.
The two most widely used heterogeneous immunoassays are the radioimmunoassay (RIA) and the enzyme linked immunosorbant assay (ELISA). In the RIA, a sample containing an unknown amount of antigen is mixed with a known amount of radiolabeled antigen and antibody. The assay components are allowed to react to near-equilibrium and then the antibodY-bound antigen is separated from the unbound antigen. Since sample antigen competes with the labeled antigen for a limited number of antibody binding sites the more antigen in the sample the less labeled antigen is in the bound fraction (or the more is in the unbound fraction). This type of assay is generally time-consuming (1-3 hours) and labor intensive.
RIA suffers from two major disadvantages: First, the labeling substance employed is a radioisotope which poses numerous problems associated with handling storage and disposal. Second, RIA is performed in a competitive mode (i.e.. the analyte and the labeled analyte compete for a limited number of binding sites on the antibody), and, therefore, the antibody affinity constant limits the sensitivity of the assay, typically in the range of 10.sup.-8 M.sup.-1 to 10.sup.-11 M.sup.-1.
ELISA is similar in principle to RIA except that the labeling substance is an enzyme rather than a radioisotope. It still suffers from the limitation that sensitivity is a strict function of the antibody affinity constant.
Other labeling substances have been described in addition to isotopes and enzymes. These include fluorophores, coenzymes bioluminescent materials and enzyme inhibitors.
Various methods of effecting the separation step in heterogeneous immunoassays are known. These include filtration, centrifugation and chromatography.
The use of affinity columns to effect the separation step has been described in French Patent Appl. No. 79 15992, published Jan. 9, 1981. It describes the use of a gel having coupled to it a ligand which has affinity for the labeling substance and which additionally has molecular sieving properties. The use of a gel having affinity for the ligand of interest rather than for the labeling substance and having molecular sieving properties is also disclosed. The assay described can be performed in a competitive or noncompetitive mode.
U.S. Pat. No. 4,298,687, issued Nov. 3, 1981 to Maes, discloses a heterogeneous immunoassay in which the substance to he determined is reacted with a labeled primary binding partner and the unreacted binding partner is then captured by absorption on a solid phase endowed with specific binding properties for the primary binding partner. The bound primary binding partner is then measured by reacting a labeled binding partner specific for the primary binding partner with the bound primary binding partner. The label is measured while still bound to the solid support. Since the primary binding partner is present in a limited amount, the reaction kinetics and equilibrium are adversely affected.
U.S. Pat. No. 3,654,090, issued April 4, 1972 to Schuurs et al., describes a noncompetitive heterogeneous immunoassay for human chorionic gonadotropin (HCG) which uses an excess of enzyme-labeled divalent antibody and an immobilized HCG column to accomplish the separation step.
U.S. Pat. No. 4.200,436, issued April 29, 1980, to Mochida et al. discloses an immunoassay employing a labeled monovalent antibody in which immobilized antigen (the same antigen as that to be measured) is used to separate the free labeled antibody from the labeled antibody-antigen complex. Since it is primarily the bound fraction which is measured this assay is usually performed in a competitive mode. Hence, sensitivity is limited by the affinity constant of the antibody when the assay is performed according to the preferred mode.
U.S. Pat. No. 4,098,876, issued July 4, 1978, to Piasio et al., discloses a reverse sandwich immunoassay in which the analyte is incubated with labeled antibody prior to incubating with the immobilized second antibody. After separation of the bound, labeled complex from the incubation medium, the bound label is measured.
U.S. Pat. No. 4,376,110, issued March 8, 1983, to David et al., discloses the use of monoclonal antibodies in a two-site sandwich immunoassay format. The preferred mode disclosed involves the measurement of the bound label after separation from the free label.
The preferred mode of operation of the heterogeneous immunoassay techniques described above is to utilize excess primary labeled binding partners and/or excess bound secondary binding partners to enhance the speed, sensitivity and precision of the assay. When operating in this preferred mode, it becomes necessary to measure the activity of the bound label since there is generally too much free label to allow accurate detections of the small decrease in the amount of free label. The detection of bound label is particularly difficult in some automated analyzers where it is often necessary to introduce the sample to the analyzer in a liquid form.
Materials such as proteins, protein-hapten conjugates and specifically antibodies can be adsorbed onto the surface of solid or liquid supports, such as polyethylene, polycarbonates, perfluorocarbon polymers, latex particles glass and magnetic particles with polystyrene being preferred. In general this adsorption is considered to be irreversible even in the presence of surfactants chaotropes, denaturants such as 8M urea or guanidine hydrochloride. [Methods in Enzymology, XL, 149, Ed. K. Mosbach, Academic Press (1976); Morrissey, B. W. Annals of the New York Academy of Sciences, 283, 50-64 (1977)].
While proteins are thought to be irreversibly bound under many aqueous conditions some solvents may cause desorption from hydrophobic surfaces. For example, dimethylsulfoxide (DMO) and tetrahydrofuran (THF) have been used in hydrophobic chromatography to desorb proteins that were adsorbed from aqueous solution onto hydrophobic supports. Presumably by changing the van der Waals or London forces between the protein and the support. These solvents, however, often attack and dissolve organic supports and. at least in the case of DMSO, denature many proteins such as enzymes. [J. Colloid and Interface Science Vol. 76, No. 1, 254-255 (1980); C. J. Van Oss et al.. Sep. Purif. Methods, Volume 7, 245 (1978); C. J. van Oss et al. Sep. Sci. Technol., Volume 14, 305 (1979)].
There exists a need for an improvement in the heterogeneous immunoassay art which has all of the advantages of the known heterogeneous immunoassay techniques but which allows rapid detection of the bound label in common automated analyzer systems.